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. 2016 Oct 25;6:36050. doi: 10.1038/srep36050

Figure 4. LMO2 blocks the canonical Wnt signaling pathway and reduces the activation of β-catenin in breast and colorectal cancer cells.

Figure 4

(A) Western blotting for β-catenin in total, cytoplasmic, and nuclear fractions of MDA-MB-231 and SW480 cells under Wnt-off and Wnt-on conditions. α-Tubulin and lamin A were used as the cytoplasmic and nuclear markers, respectively. (B) Anti-β-catenin immunofluorescence staining in MDA-MB-231 and SW480 cells under Wnt-off and Wnt-on conditions. β-catenin was stained using a rabbit anti-β-catenin antibody followed by a Fluor-568 secondary fluorescent antibody, and the nuclei were stained with DAPI. (C) Co-immunoprecipitation assays to detect the interaction between DVL-1/-2 and Axin1 in LMO2 overexpressing, control, and sh-LMO2 MDA-MB-231/SW480 cells. Cell lysates were immunoprecipitated with anti-DVL-1/2 antibodies and immunoblotted with anti-Axin1 antibodies. One milligram of total protein was used for each IP experiment and 1/20 of the total protein from each sample was used as the input. (D) The gray-scale of the immunoblotting bands corresponding to co-immunoprecipitated Axin1 was quantified using ImageJ software. The co-immunoprecipitated Axin1 values were calculated by the formula: IP-Axin1/(input-Axin1*IP-DVL-1/-2), which indicated the amount of Axin1 immunoprecipated by unit amount of DVL-1/-2 in the cell lysate containing unit amount of Axin1 in the corresponding samples. The bars represent the means of 6 independent experiments for each sample, and error bars indicate the standard error. *p < 0.05 (ANOVA followed by Dunnett’s t-test vs. control). (E) Bar plots showing the TOPflash/FOPflash reporter activity in MDA-MB-231 and SW480 cells under Wnt-off and Wnt-on conditions. The relative luciferase activity was determined after normalization to Renilla. The bars represent the means of three independent experiments, and the error bars represent the standard error. *p < 0.05 (ANOVA followed by Dunnett’s t-test vs. control). (F) Real-time PCR was used to measure c-myc, cyclin D1 and CD44 levels in MDA-MB-231 and SW480 cells under Wnt-off and Wnt-on conditions. The relative expression of each gene was normalized to the housekeeping gene GAPDH. The bars represent the means of three independent experiments, and error bars represent the standard error. *p < 0.05 (ANOVA followed by SNK-q-test).